The broad goal of this application is to understand the mechanisms by which RNA binding protein CUGBP2 regulates gene expression at the posttranscriptional level of mRNA stability and translation in intestinal epithelial cells. Overexpression of the protein in cancer cells results in cell death. We have determined that CUGBP2 interacts with AU-rich sequences in the 3'untranslated region of cyclooxygenase-2 and Mcl-1 mRNAs and upon binding downregulates the translation of both mRNAs. We have also determined that CUGBP2 levels are decreased in cancer cells but its expression is elevated in cells undergoing mitotic catastrophe. In addition, CUGBP2 is regulated at the levels of transcription by three different promoters. Activation from promoters located upstream from the commonly used proximal promoter results in the inclusion of additional amino acids in the N-terminus of the protein. Our studies suggest that the consequence of this addition is differences in cellular localization of the protein, but more importantly in the activity of the protein. The variants with the added amino acids do not affect cell viability. In addition, there are differential effects on target gene splicing with the three variants. Based on these observations, we have proposed to determine three aims. In Aim 1, we will determine the mechanism(s) by which the three CUGBP2 promoters are regulated. In addition, CUGBP2 is regulated at the posttranscriptional level of alternative splicing resulting in the novel inclusion of one intron in the 5'untranslated region. We will identify the cis-acting sequences and trans-acting factors regulating this process. In Aim 2, we will determine the cellular functions of CUGBP2. We have identified cellular factors that bind to CUGBP2. We will perform systematic deletion and fine mutagenesis to identify the domains in CUGBP2 that are involved in RNA:protein and protein:protein interactions. In addition, we will determine the CUGBP2 domains that regulate CUGBP2 localization under basal conditions and when the cells are exposed to 3-irradiation. We will also determine the effect of these interactions on CUGBP2 mediated RNA splicing, mRNA stability and translation regulation. In Aim 3, we will determine whether the CUGBP2 gene is both a novel tumor suppressor and a protooncogene. We will determine the expression levels of the three isoforms in a panel of human colon tumors. Furthermore, we will determine whether the different CUGBP2 isoforms affect tumor behavior in xenograft models. Completion of these experiments should give us a better understanding of how the RNA binding protein CUGBP2 functions in normal epithelial cells, and whether changes in the CUGBP2 expression that is observed in tumor cells is responsible for tumor behavior. PUBLIC HEALTH RELEVANCE: Colorectal cancer is the leading in cancer related deaths in the United States. Understanding how the normal cell progresses to a cancer will aid in our developing novel therapies for both diseases. We have identified a protein, CUGBP2 whose expression is lost in cancer cells. Restoring the protein into the cancer cell kills the cells. We are in the process of identifying how the gene expressing CUGBP2 is silenced in cancer cells so that we can determine ways to reverse the process. This might stop or slow down the tumorigenesis.